Blood test in health check is effective for recognizing the state of heath and early detection of a disease. In the blood test upon health check, since a large number of samples are analyzed over multiple targets, large-scaled clinical analyzers are used. Since the clinical analyzers are expensive and have to be operated by expert engineers, they are introduced in large-scaled hospitals or blood testing centers, but are not placed in general clinics. Accordingly, when blood test is performed in a general clinic, it usually takes several days for obtaining the result. This time lag causes no problem in the case of health checks since most of them are performed at a frequency of once per year or one half year. In an urgent case, however, it is necessary to conduct blood test on the spot. For example, during surgery, it is necessary to monitor blood electrolytes such as sodium, potassium, or chlorine, an oxygen partial pressure, a carbon dioxide gas partial pressure, glucose, blood urea nitrogen, hematocrit. Further, in dialysis for renal insufficiency, creatinine is measured. In addition to such urgent testing, it is also a demand for point of care testing (POCT) in order to check the health state in general clinics. The apparatus coping with such demand is a POCT apparatus, which has an advantage capable of testing on-site optionally although the number of test targets and throughputs are not so favorable as those of the clinical analyzer. The test targets include electrolyte, glucose, cholesterol, lactic acid, blood urea nitrogen, and creatinine. For general chemical measurement for glucose, cholesterol, lactic acid, blood urea nitrogen, and creatinine other than the electrolyte, an enzyme electrode method is used.
The enzyme electrode method is a method of measuring the concentration of the substance, which is converted into another substance capable of being measured by an electrode using an enzymatic reaction, indirectly by the electrode as a current or potential change. For example, in a glucose sensor for measuring a blood glucose level, glucose as a substance to be measured is oxidized by a glucose oxidase and gluconolactone is produced. By the oxidation reaction, oxygen is consumed to produce hydrogen peroxide. Since both oxygen and hydrogen peroxide are redox active compounds, the concentration of the glucose as the substance to be measured can be measured by using an oxygen electrode or a hydrogen peroxide electrode as the electrode current. However, in a case where the glucose is at a high concentration, the rate of oxidation reaction is sometimes limited by the concentration of dissolved (partial pressure) oxygen in the blood. As a countermeasure, other redox compound is sometimes used instead of oxygen. Other chemical substances can also be measured on a similar principle. Such a type of sensor is generally referred to as an amperometric enzyme sensor.
In the amperometric enzyme sensor, a working electrode, formed of gold, platinum or the like, a counter electrode and a reference electrode for keeping the potential of the working electrode constant are arranged in a solution, and an enzyme and a redox compound are in the solution. The working electrode, the counter electrode, and the reference electrode are connected to a current measuring device such as a potentiostat, such that a current value which changes upon application of a voltage between the working electrode and the counter electrode can be measured. When a sample (for example, blood) containing a substance to be measured is added to the solution, the substance is oxidized by the enzyme and, at the same time, the redox compound in the oxidized state is reduced. When a constant voltage capable of oxidizing the redox compound is applied to the working electrode, the redox compound in the reduced state is oxidized on the working electrode and a current flows in accordance with the concentration of the redox compound in the reduced state. In this way, the oxidation reaction of the substance to be measured by the enzyme can be measured as a current, and the concentration of the substance to be measured can be measured indirectly. In this case, it is necessary for an enzyme at a sufficient concentration, a redox compound at a sufficient concentration, and a working electrode of a sufficient size such that a current value in accordance with the concentration of the substance to be measured can be obtained, that is, the concentration of the substance to be measured is a rate determining factor in the reaction system.
In the amperometric enzyme sensor, an enzyme is immobilized on a membrane mainly with an aim of re-utilizing the enzyme. However, in a case where the enzyme is immobilized, since the reaction efficiency of the enzyme and the substance to be measured and the enzyme and the redox compound is lowered, the redox compound is immobilized together with the enzyme on the membrane at the surface of the working electrode (Adv. Mater. 5(1993) 912-915). It is considered that lowering of the transfer efficiency of charges from the enzyme to the redox compound can be suppressed by immobilizing the enzyme together with the redox compound on the membrane at the surface of the electrode. Further, by immobilizing the enzyme and the redox compound at a multilayer, the sensitivity is improved more and lowering of the reaction efficiency between the enzyme and the object to be measured can be suppressed compared with a case of a monolayer.
In the glucose sensor for measuring the blood glucose level, since a necessary measuring sensitivity is not so high, measuring is possible with a blood amount of several droplets. However, in a POCT apparatus for general targets, more amount of blood is necessary for maintaining the measuring sensitivity. For example, i-Stat developed as a POCT apparatus (Clin. Chem. 39/2 (1993) 283-287) required a blood amount of about 65 μl. While the blood amount can be decreased by making the electrode area smaller, since a signal (that is, current value) decreases as the electrode area is made smaller in the amperometric enzyme sensor, it was difficult to simply decrease the electrode area.
A potentiometric enzyme sensor is known as an enzyme sensor using an electric measuring method in which signals do not depend on the electrode area. The potentiometric enzyme sensor consists of a working electrode formed of gold, platinum, etc. and a reference electrode in which an enzyme and a redox compound are present in the measuring solution (JP-T No. 9-500727). Further, the working electrode and the reference electrode are connected to a device for measuring voltage. When a substance to be measured is added to the measuring solution, the substance to be measured is oxidized by an enzymatic reaction and, at the same time, a redox compound in an oxidized state is reduced. The surface potential on the working electrode generated in this case is in accordance with the following Nernst's equation.
                    E        =                              E            0                    +                                    RT                              n                ⁢                                                                  ⁢                F                                      ⁢                          ln              ⁡                              (                                                      C                    ox                                    /                                      C                    red                                                  )                                                                        [                  Formula          ⁢                                          ⁢          1                ]            whereE: surface potential of working electrode,E0: reference potential of redox compoundR: gas constantT: absolute temperaturen: difference of charges between oxidized state and reduced state of redox compoundF: Faraday constantCox: concentration of oxidized state of redox compoundCred: concentration of reduced state of redox compound
As can be seen from the equation described above, the change of the surface potential does not depend on the electrode area. Further, unlike the amperometric enzyme sensor, since a voltage is not applied to the working electrode, chemical reaction interfering the measurement less occurs. Further, since the voltage changes as the logarithm of the concentration as shown in the equation described above, a substance can be measured also in a low concentration region at an S/N ratio identical with that in a high concentration region and it is considered that a wider dynamic range can be obtained compared with the amperometric enzyme sensor.
However, in the existent potentiometric enzyme sensor, consideration is not taken on the insulative property between the working electrode and the measuring solution, and it involved a problem that actual measurement undergoes the effect of a leak current on the surface of the electrode and the sensitivity is lowered, particularly, in the low concentration region, the dynamic range narrowed and, further, the response speed is lowered.